Expression, Purification and Characterization of a Novel Hybrid Peptide CLP with Excellent Antibacterial Activity

Abstract

CLP is a novel hybrid peptide derived from CM4, LL37 and TP5, with significantly reduced hemolytic activity and increased antibacterial activity than parental antimicrobial peptides. To avoid host toxicity and obtain high-level bio-production of CLP, we established a His-tagged SUMO fusion expression system in Escherichia coli. The fusion protein can be purified using a Nickel column, cleaved by TEV protease, and further purified in flow-through of the Nickel column. As a result, the recombinant CLP with a yield of 27.56 mg/L and a purity of 93.6% was obtained. The purified CLP exhibits potent antimicrobial activity against gram+ and gram- bacteria. Furthermore, the result of propidium iodide staining and scanning electron microscopy (SEM) showed that CLP can induce the membrane permeabilization and cell death of Enterotoxigenic Escherichia coli (ETEC) K88. The analysis of thermal stability results showed that the antibacterial activity of CLP decreases slightly below 70 °C for 30 min. However, when the temperature was above 70 °C, the antibacterial activity was significantly decreased. In addition, the antibacterial activity of CLP was stable in the pH range from 4.0 to 9.0; however, when pH was below 4.0 and over 9.0, the activity of CLP decreased significantly. In the presence of various proteases, such as pepsin, papain, trypsin and proteinase K, the antibacterial activity of CLP remained above 46.2%. In summary, this study not only provides an effective strategy for high-level production of antimicrobial peptides and evaluates the interference factors that affect the biological activity of hybrid peptide CLP, but also paves the way for further exploration of the treatment of multidrug-resistant bacterial infections.

Appressoria Formation in Phytopathogenic Fungi Suppressed by Antimicrobial Peptides and Hybrid Peptides from Black Soldier Flies

“…Hoelecher et al discovered that fusion to SUMO does not appreciably affect the activity of AMPs [58]. Compared with SUMO-C-L, cecropin-like antimicrobial peptide KR12AGPWR6, and hybrid peptide CLP [57,[59][60][61], our hybrid peptide CAD-Con was also expressed normally and exhibited antifungal activities after purification.…”

The future of recombinant host defense peptides

“…Like the multidomain approach, segments (but not the full active sequence) of an HDP can be stitched together in a hybrid molecule that is a mix of the parental peptides, a process we named HDP fragment-stitching . Fragments of peptides such as the human cathelicidin LL-37, CM4 from a Chinese domestic silk moth and TP5, a fragment of the thymus hormone, have been used in one study to create hybrid antimicrobial that works against enterotoxic E. coli [ 105 ]. Fragment-stitching might be useful to remove undesired activities from a parental peptide, such as the hemolytic activity of LL-37, while generating new HDPs.…”

Yeast Expressed Hybrid Peptide CLP Abridged Pro-Inflammatory Cytokine Levels by Endotoxin Neutralization

“…Peptide hybridization is a simple and effective strategy to obtain novel AMPs with higher antibacterial and lower cytotoxicity than parent peptides [ 16 , 17 , 18 ]. CM4, LL37, and TP5 are the parent peptides for our rational design.…”

“…The superlative approach to solving this issue is by heterologous expression technology. In our previous work, we expressed a peptide termed CLP fused with a SUMO tag and demonstrated that the purified CLP had antibacterial activity [ 17 ]; however, the protein purified from bacteria still requires expensive LPS removal before clinical application. This has led us to seek other expression platforms.…”

“…In the present study, we hypothesized that the combination of CM4 [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 ], LL37 [ 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 ], and TP5 [ 1 , 2 , 3 , 4 , 5 ] may have amended LPS neutralizing and anti-inflammatory activity with the least cytotoxic and hemolytic effects. Consequently, we synthesized and expressed the hybrid peptide CLP fused with HSA in methylotrophic yeast and studied its natural activities.…”